Ribosomal protein S3: A multi-functional protein that interacts with both p53 and MDM2 through its KH domain

Sridevi Yadavilli, Lindsey D. Mayo, Maureen Higgins, Sonia Lain, Vijay Hegde, Walter A. Deutsch

Research output: Contribution to journalArticle

100 Citations (Scopus)

Abstract

The p53 protein responds to cellular stress and regulates genes involved in cell cycle, apoptosis, and DNA repair. Under normal conditions, p53 levels are kept low through MDM2-mediated ubiquitination and proteosomal degradation. In search for novel proteins that participate in this regulatory loop, we performed an MDM2 peptide pull-down assay and mass spectrometry to screen for potential interacting partners of MDM2. We identified ribosomal protein S3 (RPS3), whose interaction with MDM2, and notably p53, was further established by His and GST pull-down assays, fluorescence resonance energy transfer and an in situ proximity ligation assay. Additionally, in cells exposed to oxidative stress, p53 levels increased slightly over 24 h, whereas MDM2 levels declined after 6 h exposure, but rose over the next 18 h of exposure. Conversely, in cells exposed to oxidative stress and harboring siRNA to knockdown RPS3 expression, decreased p53 levels and loss of the E3 ubiquitin ligase domain possessed by MDM2 were observed. DNA pull-down assays using a 7,8-dihydro-8-oxoguanine duplex oligonucleotide as a substrate found that RPS3 acted as a scaffold for the additional binding of MDM2 and p53, suggesting that RPS3 interacts with important proteins involved in maintaining genomic integrity.

Original languageEnglish (US)
Pages (from-to)1215-1224
Number of pages10
JournalDNA Repair
Volume8
Issue number10
DOIs
StatePublished - Oct 2 2009

Fingerprint

Assays
Oxidative stress
Proteins
Oxidative Stress
Fluorescence Resonance Energy Transfer
Ubiquitin-Protein Ligases
Ubiquitination
DNA
Scaffolds
Oligonucleotides
DNA Repair
Small Interfering RNA
Mass spectrometry
Ligation
Mass Spectrometry
Cell Cycle
Repair
Genes
Cells
Apoptosis

Keywords

  • MDM2
  • Oxidative stress
  • p53
  • Ribosomal protein S3
  • Ubiquitination

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Ribosomal protein S3 : A multi-functional protein that interacts with both p53 and MDM2 through its KH domain. / Yadavilli, Sridevi; Mayo, Lindsey D.; Higgins, Maureen; Lain, Sonia; Hegde, Vijay; Deutsch, Walter A.

In: DNA Repair, Vol. 8, No. 10, 02.10.2009, p. 1215-1224.

Research output: Contribution to journalArticle

Yadavilli, Sridevi ; Mayo, Lindsey D. ; Higgins, Maureen ; Lain, Sonia ; Hegde, Vijay ; Deutsch, Walter A. / Ribosomal protein S3 : A multi-functional protein that interacts with both p53 and MDM2 through its KH domain. In: DNA Repair. 2009 ; Vol. 8, No. 10. pp. 1215-1224.
@article{4e890b8dd0154d648f7e76c164f918c3,
title = "Ribosomal protein S3: A multi-functional protein that interacts with both p53 and MDM2 through its KH domain",
abstract = "The p53 protein responds to cellular stress and regulates genes involved in cell cycle, apoptosis, and DNA repair. Under normal conditions, p53 levels are kept low through MDM2-mediated ubiquitination and proteosomal degradation. In search for novel proteins that participate in this regulatory loop, we performed an MDM2 peptide pull-down assay and mass spectrometry to screen for potential interacting partners of MDM2. We identified ribosomal protein S3 (RPS3), whose interaction with MDM2, and notably p53, was further established by His and GST pull-down assays, fluorescence resonance energy transfer and an in situ proximity ligation assay. Additionally, in cells exposed to oxidative stress, p53 levels increased slightly over 24 h, whereas MDM2 levels declined after 6 h exposure, but rose over the next 18 h of exposure. Conversely, in cells exposed to oxidative stress and harboring siRNA to knockdown RPS3 expression, decreased p53 levels and loss of the E3 ubiquitin ligase domain possessed by MDM2 were observed. DNA pull-down assays using a 7,8-dihydro-8-oxoguanine duplex oligonucleotide as a substrate found that RPS3 acted as a scaffold for the additional binding of MDM2 and p53, suggesting that RPS3 interacts with important proteins involved in maintaining genomic integrity.",
keywords = "MDM2, Oxidative stress, p53, Ribosomal protein S3, Ubiquitination",
author = "Sridevi Yadavilli and Mayo, {Lindsey D.} and Maureen Higgins and Sonia Lain and Vijay Hegde and Deutsch, {Walter A.}",
year = "2009",
month = "10",
day = "2",
doi = "10.1016/j.dnarep.2009.07.003",
language = "English (US)",
volume = "8",
pages = "1215--1224",
journal = "DNA Repair",
issn = "1568-7864",
publisher = "Elsevier",
number = "10",

}

TY - JOUR

T1 - Ribosomal protein S3

T2 - A multi-functional protein that interacts with both p53 and MDM2 through its KH domain

AU - Yadavilli, Sridevi

AU - Mayo, Lindsey D.

AU - Higgins, Maureen

AU - Lain, Sonia

AU - Hegde, Vijay

AU - Deutsch, Walter A.

PY - 2009/10/2

Y1 - 2009/10/2

N2 - The p53 protein responds to cellular stress and regulates genes involved in cell cycle, apoptosis, and DNA repair. Under normal conditions, p53 levels are kept low through MDM2-mediated ubiquitination and proteosomal degradation. In search for novel proteins that participate in this regulatory loop, we performed an MDM2 peptide pull-down assay and mass spectrometry to screen for potential interacting partners of MDM2. We identified ribosomal protein S3 (RPS3), whose interaction with MDM2, and notably p53, was further established by His and GST pull-down assays, fluorescence resonance energy transfer and an in situ proximity ligation assay. Additionally, in cells exposed to oxidative stress, p53 levels increased slightly over 24 h, whereas MDM2 levels declined after 6 h exposure, but rose over the next 18 h of exposure. Conversely, in cells exposed to oxidative stress and harboring siRNA to knockdown RPS3 expression, decreased p53 levels and loss of the E3 ubiquitin ligase domain possessed by MDM2 were observed. DNA pull-down assays using a 7,8-dihydro-8-oxoguanine duplex oligonucleotide as a substrate found that RPS3 acted as a scaffold for the additional binding of MDM2 and p53, suggesting that RPS3 interacts with important proteins involved in maintaining genomic integrity.

AB - The p53 protein responds to cellular stress and regulates genes involved in cell cycle, apoptosis, and DNA repair. Under normal conditions, p53 levels are kept low through MDM2-mediated ubiquitination and proteosomal degradation. In search for novel proteins that participate in this regulatory loop, we performed an MDM2 peptide pull-down assay and mass spectrometry to screen for potential interacting partners of MDM2. We identified ribosomal protein S3 (RPS3), whose interaction with MDM2, and notably p53, was further established by His and GST pull-down assays, fluorescence resonance energy transfer and an in situ proximity ligation assay. Additionally, in cells exposed to oxidative stress, p53 levels increased slightly over 24 h, whereas MDM2 levels declined after 6 h exposure, but rose over the next 18 h of exposure. Conversely, in cells exposed to oxidative stress and harboring siRNA to knockdown RPS3 expression, decreased p53 levels and loss of the E3 ubiquitin ligase domain possessed by MDM2 were observed. DNA pull-down assays using a 7,8-dihydro-8-oxoguanine duplex oligonucleotide as a substrate found that RPS3 acted as a scaffold for the additional binding of MDM2 and p53, suggesting that RPS3 interacts with important proteins involved in maintaining genomic integrity.

KW - MDM2

KW - Oxidative stress

KW - p53

KW - Ribosomal protein S3

KW - Ubiquitination

UR - http://www.scopus.com/inward/record.url?scp=70149087287&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=70149087287&partnerID=8YFLogxK

U2 - 10.1016/j.dnarep.2009.07.003

DO - 10.1016/j.dnarep.2009.07.003

M3 - Article

C2 - 19656744

AN - SCOPUS:70149087287

VL - 8

SP - 1215

EP - 1224

JO - DNA Repair

JF - DNA Repair

SN - 1568-7864

IS - 10

ER -